Abstract 2577: Pre-cancerous ovarian cells harboring a familial <i>SMARCA4</i> mutation acquire <i>in vitro</i> phenotypes associated with transformation in SSCOHT
Authors: Sonali Joshi, Shawn Campbell, Yukie Bean, Tanja Pejovic, Adam Krieg
Journal: Cancer Research (2023)

Abstract Introduction: Small Cell Carcinoma of the Ovary, Hypercalcemic Type (SCCOHT) is an extremely rare but deadly ovarian cancer associated with mutations in the SMARCA4 gene. Using a unique population of ovarian cells derived from a familial carrier of an SCCOHT-associated SMARCA4 mutation, we sought to determine if non-cancerous cells would spontaneously transform during in vitro culture, thereby providing a model system for studying formation of SCCOHT. Methods: Ovarian cells derived from a familial carrier of a SMARCA4 mutation (P590, SMARCA4. c.3081+1G&amp;gt;T) were serially passaged in vitro and interrogated for acquisition of phenotypes consistent with cellular transformation. Loss of contact-dependent inhibition was interrogated with focus-forming assays in confluent monolayers. The ability of cells to escape anoikis and grow independently as spheroids was tested in attachment-free growth conditions using both single and aggregate spheroid growth assays. Senescence-associated β-galactosidase (SA-β-gal), cellular necrosis, and cellular apoptosis was also measured. All experiments were conducted in parallel with an age-matched cell population derived from a benign ovarian surgery (P583). In experiments to determine if these differences could be attributed to loss of functional SMARCA4, non-mutant P583 cells were cultured in the presence of the SWI/SNF inhibitor BRM014 and interrogated in focus-forming and attachment-free growth assays. Results: P590 cells passaged over 12 twelve times (approx. 40 cell divisions) continued to proliferate in culture. In contrast, non-mutant P583 cells failed to grow beyond 11-12 passages. With prolonged culture, P590 cells lost expression of SMARCA4 protein, while P583 cells retained SMARCA4 expression. P590 cells grown to confluency spontaneously formed foci of multicellular aggregates, while P583 cells did not. In attachment-free culture conditions, P590 cells readily formed spheroids from both early-passage and late-passage cells. In contrast, P583 formed small spheroids that failed to expand in culture. Surprisingly, both P590 and P583 cells displayed elevated SA-β-gal staining, with neither cell populations showing elevated necrosis or apoptosis markers. Treating P583 cells with BRM014 increased focus formation and spheroid growth similar to that of mutant cells. Conclusions: SCCOHT is extremely rare, with very few representative samples useful for developing early-stage markers or improved therapeutics. Long-term culture of ovarian cells harboring inactivated forms of SMARCA4 result in the acquisition of several phenotypes consistent with early stages of cellular transformation. Our data suggest that mutating or inactivating SMARCA4 in non-transformed ovarian cells may be a way to create novel research tools for studying formation of SCCOHT. This work was supported by DoD award W81XWH-21-1-0512 Citation Format: Sonali Joshi, Shawn Campbell, Yukie Bean, Tanja Pejovic, Adam Krieg. Pre-cancerous ovarian cells harboring a familial SMARCA4 mutation acquire in vitro phenotypes associated with transformation in SSCOHT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2577.